Tuesday, 7 December 2004
14:30 - 15:00
A New Generation of Mass Spectrometric and UPLC Based Solutions for Protein Identification, Protein Expression Profiling, and Biomarker Discovery
Alistair Wallace, Waters Corporation, Manchester, UK
Proteomics is an established research discipline within academia and the biotechnology and pharmaceutical industries. Proteomics allows one to closely monitor protein expression changes in biological systems using quantitative techniques (e.g. using 2D PAGE, isotope labelling) and to subsequently identify key up- or down-regulated proteins. To that end, proteomics is a powerful technique for understanding biological pathways and disease processes, the identification of target proteins for development of future drugs and therapeutics, and more recently, the identification of disease biomarkers. Here we will address some of the key challenges that remain in the field of proteomics, in particular those faced in protein identification (qualitative) and relative protein expression profiling (quantitative).
Mass spectrometry is the method of choice for protein identification due to its speed, selectivity and sensitivity. Peptide mass fingerprinting by Matrix Assisted Laser Desorption Ionisation Time-of-flight Mass Spectrometry (MALDI-TOF-MS) is a rapid method for the identification of proteins from simple peptides mixtures, or from HPLC-separated peptide mixtures. Peptide mass fingerprinting can fail to unambiguously identify a protein if insufficient numbers of peptide ions can be detected from a parent protein. In such cases, Post Source Decay (PSD) analysis of peptide ions by MALDI-TOF, can provide the additional (sequence) specificity required to unambiguously identify those proteins. However, this approach is both sample and time consuming, as the acquisition takes several minutes for each peptide component. Here a new MALDI technique, called “parallel PSD”, will be described in which MS/MS information is acquired from all peptide ions in parallel (simultaneously), not in series. This enables all the MS/MS (sequence) data from one sample well to be acquired in just a few minutes.
Examples will be shown for the application of the parallel PSD technique to proteomics studies and will be compared with MALDI/MS/MS analysis on a Q-Tof type mass spectrometer.
Some of the common challenges faced in identifying and quantifying proteins with a classical LC/MS/MS approach will also be discussed. The use of HPLC enables extremely complex protein digests to be separated and transferred directly, via an electrospray interface, into a tandem mass spectrometer for MS/MS (sequence) analysis. “Undersampling of the proteome” is a well-documented phenomenon here, in which traditional LC-MS/MS instrumentation analyse co-eluting peptides in a serial “Data Directed Analysis” mode, resulting in a limited duty cycle. Here a novel LC/MS system will be presented which enables an MS analyser to acquire peptide data in parallel to deliver significantly enhanced sampling efficiencies and subsequent improvements in sequence coverage and quantitative information for each identified protein. In the relative quantitation of proteins, most contemporary approaches (e.g. ICAT) necessitate the chemical modification, and analysis, of only a small minority of the peptides associated with each protein contained within a sample. The LC/MS platform presented here does not require peptide derivatization, and quantitatively profiles the vast majority of the peptides within a sample to provide outstanding quantification statistics.
Examples will be shown where low abundance proteins can be detected, quantified and identified from complex biological matrices e.g. Human Serum, and where protein expression can been successfully profiled in organisms to identify key components in biological mechanisms e.g. bacterial metabolism.
